Regulation and proper timing of mitosis during the cell cycle is critical for growth an survival of an organism. The mitotic checkpoint is a pathway that determines this timing by deciding whether a particular cell is prepared to enter mitosis. Genetic screens have identified at least seven proteins in the pathway in budding yeast, however little is known of the interactions between the proteins that mediate this pathway and nothing is known about their structures. The long-term objective of this project is to understand the atomic interactions underlying the mechanism of the mitotic checkpoint and the specificity between the proteins involved. This proposal focuses on an essential element in the Saccharomyces cerevisiae checkpoint, Bub3, its structure and interaction with Bub1, a Bub3 kinase. Mammalian homologs of most of these proteins have been identified, indicating that the pathway is conserved in eukaryotes. In the course of this grant period, the following specific aims will be accomplished: 1) Determine and refine the structure of Bub3 at high resolution (less than 1:6 A). The structure will be used to understand contacts with Bub3-interacting proteins such as Bub1. It will also test the idea that WD repeats in a protein will always fold into a propeller structure. Bub3 has been cloned, expressed, purified and crystallized in this lab. A heavy atom derivative has been identified that produces peaks in isomorphous and anomoloous difference Patterson maps. 2) Locate and characterize structural features of Bub3 that are responsible for Bub1 kinetochore localization, an essential function for proper mitotic control. Crystals of Bub3 bound to Bub1 will be used to determine a structure of the complex. 3) Determine the location and structural effects of Bub3 phosphorylation, a potential regulatory event. The kinase domain of Bub1 will be expressed and used to phosphorylate Bub3. Crystals will be grown and the structure compared to the unphosphorylated protein. 4) Determine the structure of human Bub3 and compare it with the yeast structure. This structure will provide an understanding of the effects of any cancer-causing mutations. It will later be used to analyze interactions with other human mitotic checkpoint control proteins.